The goal of this research is to investigate the role of human papillomaviruses in the etiology of cervical cancer which is a significant health problem. The viral etiology of lesions that may have potential to progress to malignant lesions and the increase in sexually transmitted diseases makes it important to investigate a viral etiology for cervical carcinoma. The specific aims of this research are to determine the presence and transcriptional activity of human papillomaviruses (HPV) in benign and malignant cervical lesions, to establish an in vitro model to examine the oncogenic potential of HPV DNAs that have been molecularly cloned from genital tract lesions, and to develop immunological probes to analyze both viral and host proteins in lesions at different stages in the progression to malignancy. To examine the presence and transcriptional activity of HPV, DNA and RNA will be isolated from biopsy specimens representing stages of benign (papilloma) to malignant (invasive carcinioma) lesions. Using the Southern transfer technology to determine quantity and state (extrachromosomal/integrated), biopsy DNA will be screened with molecularly cloned probes of HPV, as well as the herpes viruses HSV-2 and CMV which have also been implicated in the etiology of cervical cancer. Quantity and quality of viral transcripts will be examined by Northern analyses, hybridization of kinase-labelled RNA to Southern transfers of viral DNA, and analysis of cDNA libraries. To establish an in vitro system to examine oncogenic potential, constructs of HPV DNA will be made with dominant selectable markers and transfected into human primary cervical epithelial (HPCE) and other recipient cells that will be assayed for phenotypic transformation and expression of HPV. The ability of HSV-2 DNA fragments to activate in trans HPV gene expression will also be examined. For manipulation and examination in vitro, cell lines of HPCE, benign, and malignant lesions will be established utilizing SV40 T antigen under the regulatory control of the metallothionein promoter. This approach provides a reversible mechanism (i.e. production of T antigen only in the presence of heavy metals) for the immortalization of epithelial cells from lesions. This will provide a source of cells that will be analyzed for molecular changes that may be relevant to malignant progression. To provide probes to investigate the presence of viral proteins and changes in host proteins in the progression from benign to malignant tumors, monoclonal antibodies will be produced to nuclear proteins of papillomata and used to screen histologically graded lesions.